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Endo-β-galactosidase digestion
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Endo-β-galactosidase digestion

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Introduction Protocol References Credit lines
Category
N-Glycans
Protocol Name

Endo-β-galactosidase digestion

Authors
Fukuda N., Michiko
Laboratory for Drug Discovery, Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST)
KeyWords
Reagents

Endo-β-galactosidase from Escherichia freundii (Amsbio, 0.1 Units/vial)

Instruments

37°C incubator

A glass column (1 × 100 cm) packed with Sephadex G-50 (superfine) and equilibrated with 0.2 M NaCl

Fraction collector

Thin layer chromatography plate (Baker HPTLC) and glass chromatography chamber

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (gel and power supply)

Scintillation counter

Methods
1.

Digestion of keratansulfates by endo-β-galactosidase2)

1) 

 Keratansulfate (3.0 mg) dissolved in 300 μL 0.2 M sodium-acetate buffer, pH 5.8.

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2) 

 Dissolve endo-β-galactosidase (0.1 Units) with 100 μL water, and add 20 μL (20 mUnits) to keratansulfate solution.

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3) 

 Incubate at 37°C for 1 h or for 48 h.

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4) 

 Apply the digest to Sephadex G-50 (superfine) column (1 × 130 cm). Collect 1.15 mL each/tube using a fraction collector and assay for hexose (Fig. 1).

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2.

Digestion of glycolipids by endo-b-galactosidase3)–5)

1) 

 Glycosphingolipid (100 μg) dissolved in 50 μL 0.2 M sodium-acetate buffer, pH 5.8, containing 200 μg sodium deoxy taurocholate.

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2) 

 Dissolve endo-β-galactosidase (0.1 Unit) with 100 μL water, and add 50 μL (5 mUnits) to glycolipid solution.

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3) 

 Incubate at 37°C for 20 h.

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4) 

 Apply the digest to thin layer chromatography plate, and develop the TLC plate in a solvent chroloform/methanol/water. TLC plate is sprayed with orcinol solution, and baked in an oven for 5 min or until purple color develops (Fig. 2).

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3.

Cell surface modification of endo-β-galactosidase6)

1) 

 Wash human red blood cells with PBS (physiological phosphate buffer) by centrifugation. Suspend red cells with an equal volume of PBS.

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2) 

 To the cells (100 μL), add 20 μL endo-β-galactosidase (20 mUnits) dissolved in PBS, and incubate at 37°C for 30 min.

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3) 

 Determine levels of carbohydrate antigens by hemagglutination using specific antibody for each blood group. Alternatively, galactose and/or N-acetylgalactosamine residues of cell surface glycoconjugates are labeled by galactose-oxidase/NaB[3H]4 followed by endo-β-galactosidase treatment. Release oligosaccharides are then analyzed by Sephadex G-50 chromatography (Fig. 3), glycoproteins are analyzed by SDS-polyacarylamide gel electophoresis (Fig. 4), and glycolipids are analyzed by thin layer chromatography (Fig. 5).

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Notes

Endo-β-galactosidase hydrolyzes linear polylactosaminoglycans, but does not hydrolyze branched polylactosamines1).  Any modification of galactose residue hinders the access of this enzyme.

Amount of enzyme required for complete digestion of the substrate depends on the structure, purity and amounts of the substrate.

Figure & Legends

Figure & Legends

 

 

 

Fig. 1. Sephadex G-50 chromatography of bovine corneal keratan sulfate digested by E. freundii endo-β-galactosidase

Chromatograms were monitored by anthrone color reaction for hexose. The smallest oligosaccahride peak IX contains GlcNAcb1-3Gal and SO4-6GlcNAcb1-3Gal.  Peak VIII contains sulfated tetrasaccahrides2).

This figure was originally published in J Biol Chem. Fukuda MN, Matsumura G. “Endo-beta-galactosidase of Escherichia freundii. Purification and endoglycosidic action on keratan sulfates, oligosaccharides, and blood group active glycoprotein” 1976, 251(20):6218–25. © the American Society for Biochemistry and Molecular Biology.  

 

 

Fig. 2. TLC of glycolipids digested by endo-β-galactosidase4)

Each purified glycolipid was digested by endo-β-galactosidase, and was separated into organic and water phases in chloroform/methanol/water (2: 1: 0.1, v/v).  Left panel shows the organic phase of the product of Ga4a (lane 1), Ga4b (lane 2) and Ga6 (lane 3), which identified glucosyl ceramide  as the product.  Right panel shows TLC of oligosaccharide products partitioned to water phase, of which structure was identified as monosialylated tetrasaccharide (1’ and 2’) and disialylated pentasaccharide (lane 3’)4).  NE1, glucosylceramide; NE2, lactosylceramide; NE3, globotriaosylceramide. TC: taurodeoxycholate (detergent).

This figure was originally published in J Biol Chem. Fukuda MN, Bothner B et al. “Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence” 1986, 261(11):5145–53. © the American Society for Biochemistry and Molecular Biology.  

 

 

Fig. 3. Gel filtration analysis of oligosaccahrides released by endo-β-galactosidase from cell surface labeled human erythrocytes

Human erythrocytes were labeled by galactose oxidase/NaB[3H]4 treatment. Erythrocytes were then treated with endo-β-galactosidase and released oligosaccahrides were applied to Sephadex G-50 column. Radioactivity in each fraction was counted by a scintillation counter. Open circle: released radioactivity after endo-β-galactosidase treatment; closed circle, released radioactivity by control treatment. This figure was originally published in J Biol Chem. Fukuda MN et al. “Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes” 1979, 254(12):5458–65. © the American Society for Biochemistry and Molecular Biology.  

 

 

  Fig. 4. SDS-PAGE of surface labeled human red blood cells treated with or without endo-β-galactosidase6)

Lanes 1 and 2 are Commassie blue staining of the gel showing erythrocyte membrane proteins.  Band 3 (anion transporter) and band 4.5 (glucose transporter) are heterogeneously glycosylated by polylactosamines, giving broad bands. Lane 3 and 4 are fluorogram of cell surface labeled erythrocyte membranes after treatment without (lane 3) or with endo-β-galactosidase (lane 4).  Note that radioactivity associated with band 3 and band 4.5 disappeared after endo-β-galactosidase digestion.

This figure was originally published in J Biol Chem. Fukuda MN et al. “Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes” 1979, 254(12):5458–65. © the American Society for Biochemistry and Molecular Biology.  

 

 

 

 

Fig. 5. Fluorogram of TLC analysis of cell surface labeled erythrocyte glycolipids6)

Glycolipids were extracted from the membranes of cell surface labeled human erythrocytes treated with (lane 2) or without (lane 1) endo-β-galactosidases.  Radioactive glycolipids were separated by TLC and visualized by fluorography.  Note that endo-β-galactosidase removed labels at H2, H3 and H4 regions, which are made of polylactosaminyl structure, whereas short lacto-series glycolipids (H1) or globo-series (Glo) structure were not susceptible to endo-β-galactosidase. 

This figure was originally published in J Biol Chem. Fukuda MN et al. “Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes” 1979, 254(12):5458–65. © the American Society for Biochemistry and Molecular Biology.

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